The expression of wild type and mutant histone H3 and H4 genes will be studied in the yeast Saccharomyces cerevisiae by techniques of molecular genetics. The genes have been cloned, sequenced, and their mRNAs have been mapped. Histones H3 and H4 are expressed during a brief time in the cell division cycle at or before DNA replication. How is this expression regulated? The experiments will test the hypothesis that events at an origin of DNA replication, closely linked to the histone genes, induce a periodic local change in chromatin conformation and that this permits derepression of histone transcription. Using the cloned H3 and H4 genes, transcription will be assayed in cell division cycle mutants to determine the timing and relationship of transcription to other cell events. Rates of transcription will be measured by selection of pulse-labeled mRNA by hybridization with M13 single stranded DNA gene probes. Cell cycle changes in chromatin conformation will be studied by nuclease sensitivity assays. The intergene DNA containing the histone promotor/activator sequences will be analyzed by deletion and linker-scanning mutagenesis. Levels of expression will be assayed using gene fusions of the manipulated histone promotor regions with bacterial Beta-galactosidase. The histones H3 and H4 are key components of the nucleosome, the fundamental unit of chromatin organization. What is the genetics of histone function? Mutations in the histone H4 molecule will be constructed using techniques of in vitro site directed mutagenesis. Mutations will be identified by DNA sequence analysis. Plasmid vectors carrying the mutant H4 genes will be introduced into yeast by DNA transformation. They will be tested for the ability to transform, and the genes will be examined for transcription, translation, and the effect of the mutant H4 proteins on cell phenotype, including possible conditional leathals. The proposed experiments are relevent to questions of cell division cycle regulation, control of cell proliferation, and chromatin conformation and its role in regulating gene activity.